The preparation of polypeptides using recombinant technology has developed into a standard procedure during the last couple of decades. The access to recombinant polypeptides by cloning the genes encoding the respective polypeptide followed by subsequent transformation of suitable expression hosts with the gene to be expressed and final production and purification of the obtained recombinant polypeptide product has provided access to a whole new class of biologically designed and produced therapeutics.
Pharmaceutically active compounds have been prepared in increasing numbers in the pharmaceutical industry using recombinant DNA technology followed by production processes developed in the field of bioengineering.
Such biological products include monoclonal antibodies, which have been developed into important treatment options in various medical fields including autoimmune diseases, inflammatory disorders, immunosuppression, oncology or the like.
Development of such therapeutics of biological origin requires production at industrial scale thereby providing access to large amounts of recombinant polypeptide. Preferred expression systems are mammalian cell cultures which are superior to most other eukaryotic systems based on insect cells, yeast or the like, or even traditional prokaryotic expression systems.
However, mammalian cell culture includes tremendous challenges especially at the industrial scale. Production facilities for mammalian cell culture require thorough optimization of many process conditions.
One of the most important process parameters for controlling the overall production process is the medium in which cells are grown. Suitable cell culture media must provide cell cultures with all necessary nutrients, which is especially difficult if no components of animal origin like serum or proteins, e.g. growth factors, are added to the media.
Further, mammalian cell cultures require particular supplement components at different stages of the polypeptide production process. Accordingly, cell culture media must provide the necessary substrates during a) initial growth and proliferation of the host cells at lower densities; b) subsequent cultivation of cells to high densities, c) the actual process of polypeptide formation in the cultured cells.
The overall process for the production of recombinant polypeptide preferably comprises an expansion phase and a production phase. During expansion phase the host cells are cultivated to high densities by using a growth medium in order to maximize subsequent polypeptide production later on During production phase the actual formation of the desired polypeptide in large amounts is achieved by use of a production medium. In order to meet the specific metabolic requirements of the cells in each phase of the overall polypeptide production process, different media compositions have been designed for expansion and production phase respectively. For instance, production media often contain higher amounts of amino acids than growth media.
Accordingly, considerable efforts have been taken in the past to develop cell culture media with special emphasis on their use for large scale production of polypeptides. Nevertheless, continuous improvement of cell culture media is still an important goal in order to further maximize polypeptide production in terms of product quality and quantitative yields.
Many components of cell culture media have been investigated in the past in terms of their role for polypeptide production. Possible targets are inorganic salts, amino acids, sources of carbon like glucose, or vitamins.
For instance, it has been demonstrated that supplementation of compounds like vitamins, choline chloride or amino acids can increase viability and productivity of cells cultivated under protein-free conditions (Kim do Y et, al., Cytotechnology 2005, 47, 37-49).
Choline chloride is a standard component of cell culture media which serves as a phospholipid precursor for the cells. After being taken up and being processed by the cells, it ends up, besides phosphatidylethanolamine and phosphatidylinositol as one of the major phospholipids in cell membranes called phosphatidyl choline.
Commonly used cell culture media like D-MEM (Dulbecco's Modified Eagle Medium) and D-MEM/F-12 have been widely used for the growth of a wide range of mammalian cell lines. These media include amounts of choline chloride of 4 mg/L and 8.98 mg/L, respectively.
Other commercially available media like Ham's F-12 (commercially available from BioConcept) and MEM (commercially available form HyClone) also comprise low amounts of choline chloride of 13.96 mg/L and 56 mg/L, respectively.
U.S. Pat. No. 6,180,401 discloses an improved method for producing a polypeptide in animal cell culture. One objective is to increase the final product concentration. Several parameters are modified in order to maximize product yield in the production phase including glucose concentration, osmolality and glutamine concentration. U.S. Pat. No. 6,180,401 discloses cell culture media, which have a content of choline chloride of 50.86 mg/L.
U.S. Pat. No. 5,122,469 discloses a culture medium for propagating various mammalian cell lines, in particular Chinese hamster ovary cells (CHO), and allows the cultivation of cells at high densities as monolayers or in suspension suitable for small and large scale propagation of mammalian cells. One further advantage is an enhanced yield of product. The medium is a chemically defined culture medium containing elevated levels of certain amino acids. The content of choline chloride is 50.86 mg/L.
Only very few media with high content of choline chloride are known in the prior art. Waymouth has described a cell culture medium, which can be used for the culture of the mouse L929 fibroblast connective tissue cell line (C. Waymouth, J. Natl. Cancer. Inst., 1959, 22, 1003-1017). This medium is a serum-free, chemically defined synthetic medium and has a content of choline chloride of 250 mg/L. This medium is commercially available under the name Waymouth's Medium MB 752/1 (BioConcept and Sigma-Aldrich). Known applicability is limited to whole organ culture, establishment of carcinoma cell lines from pleural effusions, and the growth of potentially tumorigenic cells prior to their assessment in vivo.
WO 02/101019 discloses two medium compositions with relatively high content of choline chloride, 101.72 mg/L and 209.40 mg/L, respectively. These media were used for studying the impact of glutamine and glutamate for recombinant protein production. However, both media still contained high amounts of glutamine.
Only limited information is available from the prior art as far as the role of the choline chloride content in cell culture media for polypeptide production is concerned. U.S. Pat. No. 6,048,728 briefly discusses the role of choline chloride content in cell culture media for the production of biological products using hybridoma cells. In the case of antibody expressing cells, secretion of maximum amounts of antibody was observed in media containing a choline supplement of greater than 4 mg/L and preferably of approximately 4 to 75 mg/L, in combination with the other reagents of the Primary Supplement. At these concentrations, choline is described to be not limiting and being without apparent toxicity.
Production cell culture media, especially those designed for use in industrial large scale production of recombinant polypeptides requires increased amounts of components, for instance amino acids.
However, highly concentrated cell culture media show limited solubility of selected media components. Limited solubility represents a technical disadvantage because highly concentrated media for large scale production bear the risk of precipitation of individual components, for instance during the production phase and especially during storage. This can lead to variations of the media composition and to a deterioration of the cell culture conditions at the critical point of product formation.
As a further consequence, precipitation leads to the effective removal of precious media components from the actual production process. Additional recycling processes designed for overcoming such drawbacks are technically difficult to realize and require further effort in terms of resources and time Less concentrated cell culture media, when equally effective in polypeptide production, would allow achieving significant cost reductions in industrial production processes.
Considering the above challenges and existing disadvantages, there is a continued need in the field of industrial biotechnology for improved culture media which allow producing recombinant polypeptides at an industrial scale with even higher yields, i.e. improved specific and overall productivity, and increased product quality. Improved cell culture media are especially desirable for improvement of productivity during production phase.
A specific technical objective of polypeptide production processes is to maintain higher cell viabilities at the end of the production process in order to maximize the final yield of polypeptide in particular due to prolongation of the production time. Moreover, reducing the aggregation of the formed recombinant polypeptide and improved product quality particularly in terms of posttranslational modifications, such as glycosylation pattern is also an important technical objective.
Finally, improved production media for large scale production of polypeptides are desirable which contain reduced amounts of components while being equally effective or even better in terms of cell growth, polypeptide productivity, recombinant polypeptide quality and polypeptide functionality.